The incorporation of 32P into spectrin aggregates following incubation of erythrocytes in 32P-labelled inorganic phosphate

³²P was incorporated into spectrin by incubation of fresh erythrocytes with ³²P_i and glucose. The dimer and tetramer aggregates revealed only covalently-bound incorporation of phosphorus, while a higher aggregate of spectrin revealed both covalent and non-covalent incorporation. The specific activity of the covalently-bound phosphorus in all oligomers was identical, suggesting that the state of association is independent of phosphorylation. The non-covalent incorporation was shown to be due to the association of ATP with this higher aggregate. The nucleotide appears not to be bound directly to spectrin but rather to component 5 (erythrocyte actin) which is also found to be associated with this highly aggregated spectrin structure.     

Effect of different soil and nitrogen fertilizer conditions on 32P-labelled phosphate uptake by two grass species

In two years of trials, roots of ryegrass took up more ³²P-labelled phosphate than roots of fescue. Application of 672 kg N ha^-1 increased phosphate absorption compared with application of 112 kg N ha^-1. Roots in mineral soil absorbed more phosphate than those in peat soil. In both soils uptake decreased as depth of phosphate injection increased from 5 to 30 cm. An interaction occurred whereby roots in the intermediate depth (10–22-5 cm) in peat absorbed less phosphate than in mineral soil and this was apparently unrelated to the exchange or sorption properties of the soil.     

The lack of direct coupling between ATP-ADP translocase and creatine phosphokinase in isolated rabbit heart mitochondria

The formation of creatine phosphate by isolated rabbit heart mitochondria in the presence of creatine, α-ketoglutarate, ATP, and inorganic phosphate was studied. Creatine phosphate formation was inhibited by oligomycin. This was most probably due to increased concentration of ADP favoring the reverse reaction (formation of creatine and ATP from phosphocreatine and ADP). The inhibitory effect of oligomycin disappeared in the presence of phosphoenolpyruvate and pyruvate kinase. The results do not indicate any direct coupling between mitochondrial creatine phosphokinase and ATP-ADP translocase as has been suggested for rat heart mitochondria.     

Direct effect of insulin on the labeling of isolated plasma membranes by [γ32P] ATP

Labeling of isolated plasma membranes from rat adipocytes by [γ³²P] ATP was studied and the effect of insulin on this process was determined. Labeling was bi-phasic, increasing to a maximum while the supply of ATP was sufficient, then decreasing as the supply of ATP was depleted by hydrolysis. Insulin added directly to the assay mixture without pre-incubation decreased the level of labeling of plasma membranes at each time of incubation tested, from 30 sec to 10 min. The effect was insulin dose-dependent and was not observed with the inactive insulin derivative, desoctapeptide-insulin.     

Interaction of creatine kinase with phosphorylating rabbit heart mitochondria and mitoplasts

This paper demonstrates that the mitochondrial isoenzyme of creatine kinase (CKm) can be solubilized from rabbit heart mitochondria, the outer membrane of which has been removed or at least broken by a digitonin treatment or a short hypotonic exposure, but which has retained an important part of the capacity to phosphorylate ADP. Phosphate, ADP, or ATP, at concentrations which are used to study oxidative phosphorylation and creatine phosphate synthesis, solubilize CKm; the same is true with MgCl2 and KCl. The effect of adenine nucleotides does not seem to be due to their interaction with the adenine nucleotide translocase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that CKm is the main protein released in the described conditions; however, it does not amount to more than 1% of the total protein content of the mitoplasts. When the apparent Km for ATP of CKm was estimated by measuring creatine phosphate synthesis, the values obtained using water-treated mitochondria (0.21 mM) were slightly higher than those of intact mitochondria (0.12 mM) but the difference was not significant. In the former preparation 77% of CKm was in a soluble state. If we can extrapolate these results to intact mitochondria and suppose that in this case a fraction of CKm is also soluble in the intermembrane space, this does not support the theory of functional association between CKm and the adenine nucleotide translocase.     

The reactivity of -SH groups in the ADP/ATP carrier isolated from beef heart mitochondria

The emergence of the reactivity of -SH groups associated with conformation changes has been studied on the ADP/ATP carrier, is isolated in three different inhibitor-protein complexes. 1. The bongkrekate-protein complex incorporates approximately one molecular more of N-ethylmaleimide than the carboxyatractylate-protein complex. After extensive denaturation by dodecylsulfate in urea, both inhibitor complexes exhibit four reactive -SH groups per subunit. Thus one of four -SH groups per subunit has been unmasked in the bongkrekate-protein complex. 2. The interconversion from the bongkrekate-protein complex to the carboxyatractylate-protein complex is inhibited after the -SH groups have been blocked. 3. The protein complex isolated with the more easily dissociable atractylate, is used to demonstrate, by the emergence of the -SH groups, the transition into the m-state. This transition is specifically catalyzed by ADP and ATP. 4. Using 2,2'-dinitro-5,5'-dithiodibenzoate, the appearance of the -SH groups on transition from the c-state to the m-state can be followed spectrophotometrically. The specificity for the catalyzing nucleotides is identical with that for the transport. The Km for ADP and ATP is in the range of 1 microM. In conclusion, the thiol groups of the isolated ADP/ATP carrier behave as in the mitochondrial membrane. The unmasking of -SH groups is in full accordance with the concept of two conformational states (c and m).     

A 31P-NMR study of the cross-membrane pH gradient induced by ATP hydrolysis in mitochondria

³¹P-NMR has been used to study the increase of ΔpH in mitochondria by externally added ATP. Freshly prepared mitochondria was treated with N-ethylmaleimide to inhibit the exchange between internal and external P_i. Upon addition of ATP, phosphocreatine (30 mM) and creatine kinase to a NMR sample of mitochondria suspension (approx. 120 mg protein/ml) at 0°C, an increase of ΔpH by approx. 0.5 pH unit was observed. However the increased ΔpH could not be maintained, but slowly decayed along with the increase of external ADP/ATP ratio. Further addition of valinomycin to the suspension induced a larger ΔpH (approx. 1) which was maintained by the increased rate of internal ATP hydrolysis as seen in the growth of the internal P_i peak intensity in NMR spectra and the concomitant decrease of the external phosphocreatine peak. The external P_i and ATP peaks stayed virtually constant. When carboxyatractyloside was added to inhibit the ATP/ADP translocase, the internal P_i increase was stopped and the ΔpH decayed. These observations in conjunction with those made earlier in respiring mitochondria clearly show the reversible nature of the ATPase function in which the internal ATP hydrolysis is associated with outward pumping of protons.     

The effect of inorganic phosphate on creatine kinase in respiring rat heart mitochondria

The ability of creatine to stimulate the respiration of rat heart mitochondria in vitro is reversibly affected by the concentration of inorganic phosphate. The rate of oxygen consumption due to post-ADP state-4 respiration in the presence of 20 mm creatine is reduced significantly when the potassium phosphate concentration is raised from 5 to 20 mm. State-3 respiration is reduced only by potassium phosphate concentrations higher than 20 mm. The rate of synthesis of creatine phosphate is also affected by phosphate concentration, and the apparent K_m of the coupled reactions for ADP is significantly higher at 25 mm phosphate as compared to that at 5 mm phosphate. These observations are consistent with the hypothesis that inorganic phosphate acts as an effector molecule, regulating creatine phosphate synthesis by favoring the dissociation of mitochondrial creatine kinase from the mitochondrial membrane. Such regulation may be important in the case of cells undergoing partial or severe ischemia, where changes in phosphate concentration within this range have been reported.     

Purification of creatine kinase from beef heart mitochondria]

53-fold purified creatine kinase is isolated from beef heart mitochondria by phosphate buffer extraction followed by chromatography on DEAE-cellulose and KM-cellulose and preparative electrophoresis in phosphate buffer density gradient. The purified enzyme was homogenous under electrophoresis in agarose gel and moved to cathode. The enzyme did not enter into separating gel under disc electrophoresis in conditions for the separation of neutral anc acid proteins, while under conditions for separating alkaline proteins it produced five fractions. The stability of creatine kinase under storage considerably decreased after the purification.     

Iron transport by proteoliposomes containing mitochondrial F1Fo ATP synthase isolated from rat heart

In this work, we present evidence of Fe²⁺ transport by rat heart mitochondrial F₁F_o ATP synthase. Iron uptake by the vesicles containing the enzyme was concentration- and temperature-dependent, with an optimum temperature of 37 °C. Both ATP and ADP stimulated iron uptake in a concentration-dependent manner, whereas AMP, AMPPCP, and mADP did not. Inhibitors of the enzyme, oligomycin, and resveratrol similarly blocked iron transport. The iron uptake was confirmed by inhibition using specific antibodies against the α, β, and c subunits of the enzyme. Interestingly, slight transport of common divalent and trivalent metal ions such as Mg⁺², Ca⁺², Mn⁺², Zn⁺², Cu⁺², Fe⁺³, and Al⁺³ was observed. Moreover, Cu⁺², even in the nM range, inhibited iron uptake and attained maximum inhibition of approximately 56%. Inorganic phosphate (Pi) in the medium exerted an opposite effect depending on the type of adenosine nucleotide, which was suppressed with ATP, but enhanced with ADP. A similarly stimulating effect of ATP and ADP with an inverse effect of Pi suggests that the activity of ATPase and ATP synthase may be associated with iron uptake in a different manner, probably via antiport of H⁺.     

Dissociation and reassociation of creatine kinase with heart mitochondria; pH and phosphate dependence.

Experimental evidence is given of mitochondrial creatine kinase ability to dissociate from or reassociate with mitochondrial membrane as compared to the behaviour of adenylate kinase. CK release occurs for P_i concentrations higher than 5 mM and is strongly pH-dependant. Solubilized CK is able to reassociate with mitochondrial inner membrane when either P_i concentration or pH are decreased. The possible physiological effects of events, such as ischemia, which modify the intracellular pH or P_i concentration are discussed, in view of the special role which has been attributed to mitochondrial CK in the transfer of energy in heart cells.     

Essential arginine residues of creatine kinase from beef heart mitochondria

Creatine kinase from beef heart mitochondria is inactivated by 2,3-butanedione. The kinetics of inactivation of the mitochondrial enzyme is biphasic with a bend at a point corresponding to 50% inactivation. The inactivation rate constants of the first fast and the second slow phases of the reaction differ by one order of magnitude, thus suggesting the existence of two types of arginine residues, i.e. "fast" and "slow" ones, with different reactivities. The inactivation rate constant of the slow phase is very close to that for cytoplasmic creatine kinase. At saturating concentrations MgATP and MgADP afford complete protection of the slow phase of inactivation. It is assumed that the "slow" arginine is involved in the binding of metal-nucleotide substrates in the enzyme active center.     

Metabolic characteristics of mitochondria isolated from rabbit brain

Abstract— Data on the metabolic characteristics of mitochondria from rabbit brain have been obtained using low concentrations (100–300 μm) of ¹⁴C-labelled substrates of the Krebs tricarboxylic acid cycle. Oxygen consumption was measured polarographically with a Clark-type electrode. Products of the reactions were separated by chromatography on Dowex-1 columns. Data are reported for relative respiratory quotients of various substrates, respiratory control ratios, accumulation of ¹⁴C in various tricarboxylic acid cycle intermediates and the effect of malate and several inhibitors on these parameters. The data have been compared with similar experiments on mitochondria from rabbit heart. Mitochondria from rabbit brain differ substantially from heart mitochondria: those from brain have a very low capacity for oxidation of acetate and other short chain fatty acids; they do not form j3-hydroxybutyrate; they produce substantial amounts of α-ketoglutarate and require added dicarboxylic acid to yield a maximum respiratory quotient. Like those from heart, brain mitochondria have a high capacity for pyruvate oxidation and a low K_m for this substrate. The results have been discussed and compared with work reported by other investigators.